NUSAP1 Promotes Tumor Growth And Indicates Poor Prognosis In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma(HCC)is one of the most common maligancies that threatens the health and life of human beings.It is the sixth leading cause of malignancy with high morality.At present,Surgical treatment is the main treatment for HCC.Although there are many treatments for hepatocellular carcinoma,the majority of patients still inevitably die from tumor recurrence and metastasis.However,the exact molecular mechanism is still not entirely clear.Therefore,further exploration and study on the relationship between the new gene function and features of HCC is of great significance to reveal the exact molecular mechanism,design a reasonable treatment scheme and judge the prognosis of HCC.Nucleolus spindle-related protein(NuSAP1)is an important microtubule-binding protein,which is involved in microtubule crosslinking during mitosis,regulates the function of centromeric microtubules,and governs the oscillation of chromosomes.In addition,NUSAP1 regulates spindle assembly,chromosome separation,and cell division.In recent years,studies have shown that high expression of NUSAP1 exists in pancreatic cancer,melanoma,glioblastoma,acute lymphoblastic leukemia,breast cancer,acute myelogenous leukemia and many other tumors,but the exact mechanism involved in cell biology between NUSAP1 and hepatocellular carcinoma are not very clear.Our research is aim to explore the expression of NUSAP1 in HCC and its relationship with poor prognosis.Furthere more,we also investigate the biological role and molecular mechanism of NUSAP1 in the development of HCC,and search for molecular markers and drug targets related to early diagnosis of HCC.Methods:1.We performed a meta-analysis of available transcriptome data from 6independent HCC datasets [5 datasets from the Gene Expression Omnibus(GEO)and1 dataset from The Cancer Genome Atlas(TCGA)] to identify the differentiallyidentified genes(DEGs).2.Clinical data from TCGA were extracted and the data were analyzed to investigate the relationship between NUSAP1 and clinicopathological classification of HCC and the Prognosis of patient.3.Rna-seq data of HepG2_Control and HepG2_shNUSAP1 cells were used for differential expression analysis and pathway analysis.4.Two representative HCC cell lines were used to construct a cell model of NUSAP1 knockout.After transfection for 48 h,the expression level of NUSAP1 was tested by qRT-pcr to evaluate the silencing effect of NUSAP1.5.ShNUSAP1,shNC and shFoxM1 were transfected into two liver cancer cell lines,Huh7 and SMCC-7721,respectively.After 48 hours,cell proliferation rate was detected.6.SMCC-7721 cell lines were transfected with sh-NUSAP1-2 and sh-NUSAP1-3 for colony forming experiment,Quantitative analysis was performed using ImageJ software7.SMMC-7721 cells transfected with sh-NC and sh-NUSAP1 plasmids for 48 h were subjected to cell cycle assays.Result:1.Analysis based on GEO and TCGA databases showed that NUSAP1 expression up-regulated in tumor tissues compared to non-tumor tissue.2.The overall survival curve showed that high NUSAP1 expression leads to poor prognosis,while low NUSAP1 expression patients have longer survival time;The clinical data implied that high NUSAP1 expression was associated with tumor histologic grade.3.Pathway analysis based on RNAseq data suggested that NUSAP1 could active the expression of genes involves in cell proliferation.4.sh-NUSAP1-2 could decrease the expression level of NUSAP1 to 11%,so sh-NUSAP1-2 is used for follow-up experiments.5.the cell proliferation in Huh7 and SMMC-7721 cell lines was inhibited dramatically,compared to the shCtrl groups and the difference was statisticallysignificant(Huh7: P= 0.019;Smmc-7721: P=0.006).6.The results of colony forming experiment showed that the growth of SMMC-7721 cells was significantly inhibited by NUSAP1 knockdown.7.NUSAP1 knockdown inhibited cell cycle progression and arrested most cells at the G1 phase.Conclusion:In conclusion,compared to that in nontumor tissues,NUSAP1 is highly expressed in HCC tumor tissues.Analysis of clinical data showed that HCC patients with high NUSAP1 expression have poor prognosis.In addition,the results of cell proliferation assays also proved that NUSAP1 plays a crucial role in HCC cell proliferation.Our findings indicate that NUSAP1 might be a novel diagnostic marker and a therapeutic target in HCC.